Methods And Compositions For Growth Of Hydrocarbons In Botryococcus sp.

ABSTRACT

Acceleration of botryococcenoids and growth by concomitant provision of appropriate light, minerals, and assimilable carbon. Specifically, methods, compositions and systems for the in vitro growth of hydrocarbons in photosynthetic organisms while maintaining a biologically exclusive monocultural environment, as for example, from  Botryococcus  species, is disclosed. Niche-nutrients can include about 200 ppm to about 3% nitrogen, and about 100 ppm to about 15% P 2 0 5 , and about 100 ppm to about 3.5% K 2 0. In certain embodiments, the present invention relates to the growth of the Chlorophyta such as  Botryococcus  sp. in a nutrient medium that includes up to 15% phosphates, at least 3 ppm soluble iron, and up to about 70 ppm soluble zinc. Also disclosed is a substantially pure culture of  Botryococcus braunii  var.  Showa , strain Ninsei, having the ATCC Accession No. PTA-7441, its parts, and hydrocarbons produced therefrom.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. Ser. No. 11/429,536 filed May5, 2006, which claims priority of provisional application Ser. No.60/678,711, filed May 6, 2005, the disclosures of which are herebyincorporated by reference. This application is related to Plant PatentNo. PP21091 issued Jun. 22, 2010, the disclosure of which is herebyincorporated by reference.

BACKGROUND OF THE INVENTION

The present invention is a novel and distinct process for commercialgrowth of hydrocarbons in photosynthetic organisms while maintaining abiologically exclusive monocultural environment, as for example, in thecase of the present invention, from Chlorophyta, Trebouxiophyceae, andparticularly Botryococcus species.

For decades, Botryococcus species have been suggested as potentialsources of liquid transport fuels (Wolf, et al. 1985). Academically,Botryococcus species have proven quite attractive for their naturalchemistries, but the cost for production of a gallon of renewabletransport fuel exceeded the sales price of fossil fuels. A number ofdifferent culture conditions have been investigated, but a definedsystem for competitive growth of transport fuels has not been disclosedpreviously. Humanity would benefit from development of a reliable systemfor production of petroleum-type hydrocarbons from a renewable energysource.

Botryococcus is a primitive colonial photosynthetic organism, datingfrom 300 million years ago, and may be regarded as a living fossil; as,indeed, B. balkachicus; B. coorongianus; B. luteus; and B. palanaensis,are true fossil deposits. Oil shale is populated with botryococcitefossils from which petroleum deposits arose. Shale originates from mud,and in consideration of the fossil record, the methods, compositions andorganisms of the present invention, allow for expression of the mudorigins of live Botryococcus species, including the following: B.australis, B. braunii, B. braunii var. horridus, B. braunii var. minor,B. braunii var. perarmatus, B. braunii var. Showa, B. braunii var.validus, B. calcareous, B. canadensis, B. comperei, B. fernandoi, B.giganteus, B. micromorus, B. neglectus, and B. pila. As a result ofdefining its proper niche in the course of the present invention, rapidgrowth at the water-to-air interface was made possible by elimination ofbiological competition.

At the surface of mud, water evaporates and salts become concentrated tothe extent that crystals may accumulate at the surface. In the presentinvention, I have discovered that, consistent with a mud niche, aflotation mechanism related to hydrocarbon metabolism may have evolvedin photosynthetic organisms to utilize the concentrated nutrient saltsat moist surfaces. That is, through natural selection, Botryococcus sp.became one of the most successful photosynthetic eukaryotes on Earth bysurvival in an environment intolerable to competitors. In the presentinvention, the environmental tolerances that made Botryococcus sp. thefittest for hundreds of millions of years are defined and utilized innovel systems for growing hydrocarbons such as gasoline commercially.Based on this understanding of Darwin's concept of, “survival of thefittest,” oleomic photosynthetic organisms were tested in nutrient saltsat very high concentrations. Surprisingly, Botryococcus sp. thrived inhundreds times the concentrations of the salts in conventionalnutrients. The present invention exploits the aforementioned discoveryof the exclusive niche of Botryococcus sp.

SUMMARY OF THE INVENTION

The present invention relates to methods, compositions and systems forthe in vitro growth of hydrocarbons in photosynthetic organisms whilemaintaining a biologically exclusive monocultural environment, as forexample, from Botryococcus species. The environmental tolerances thatmade Botryococcus sp. the fittest for hundreds of millions of years aredefined and utilized in novel systems for growing gasoline on acommercial scale. The present inventor discovered that Botryococcus sp.thrived in many times the concentrations of the salts in conventionalnutrients; it out-competes all other life forms by living in achemically extreme environment of high concentrations of all of itsnutrient chemicals. The present invention exploits the aforementioneddiscovery of the exclusive niche of Botryococcus sp.

In certain embodiments, niche-nutrients include about 200 ppm to about3% nitrogen, and about 100 ppm to about 15% P₂0₅, and about 100 ppm toabout 3.5% K₂0. In certain embodiments, the nutrient medium is abalanced nutrient salt formulary comprising phosphate salts andincluding ammonium salts, calcium salts, potassium salts, magnesiumsalts, sodium salts, phosphoric acid, pyrophosphates, polyphosphates,glycerophosphates, and the like; with soluble potassium phosphates, mosthighly preferred.

In certain embodiments, the present invention relates to the growth ofthe Chlorophyta such as Botryococcus sp. in a nutrient medium thatincludes about 800 ppm to 15% phosphates and about 2 ppm to about 70 ppmsoluble zinc.

In certain embodiments, the present invention relates to the growth ofphotosynthetic organisms such as Botryococcus sp. in a nutrient mediumthat includes soluble iron, manganese and magnesium at concentrationsfar greater than conventional phycological nutrients in order to furtherenhance synthesis of hydrocarbons.

In certain embodiments, the present invention relates to the growth ofthe Chlorophyta such as Botryococcus sp. in a nutrient medium thatincludes phosphate salts, including, ammonium salts, calcium salts,potassium salts, magnesium salts, sodium salts, phosphoric acid,pyrophosphates, polyphosphates, glycerophosphates, and the like,phosphate buffers comprised of monobasic, dibasic, and tribasic salts;citrates; Krebs Cycle carboxylates; and derivatives thereof and thelike. Suitable ranges of nutrients include 0.800 ppm to 30% phosphates,with preferred phosphate concentration of from about 0.800 ppm to about3% phosphates, about 25 ppm to about 250 ppm soluble magnesium, about0.3 ppm to about 3 ppm soluble manganese, about 0.3 ppm to about 10 ppmsoluble iron, preferably about 5 ppm to about 9 ppm soluble iron, mostpreferably about 6 ppm to 8 ppm soluble iron, and about 0.2 ppm to about70 ppm soluble zinc (Zn⁺²).

The present invention also relates to a substantially pure culture ofBotryococcus braunii var. Showa, strain Ninsei, having the ATCCAccession No. PTA-7441, its parts, and hydrocarbons produced therefrom.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the meniscus at the top of a clear glass culture cylinderthat is highly populated with colonies of Botryococcus sp. afloat at thesurface and in the thin film of water being drawn above the meniscus bycapillary action of the mass aggregate of colonies. The culture waslightly tapped just prior to photography to release some colonies intothe water below the meniscus to display separated colonies as best aspossible, and, as a result, the colonies followed a sine wave patterninto submergence just below the surface. All colonies that weresubmerged by the wave, floated back again to the meniscus. The unit ofscale to the right is in millimeters. The colonies can be cultured onany water-moistened solid surface that can hold and supply nutrients inan aqueous medium, including, paper, plastic, colloids, phycocolloids,agar, agarose, carrageenan, agar substitutes, textiles, gel, mud, soil,earth, shale, clays, foam, ceramics, concrete, brick, metal foam,wovens, nonwovens, and solid and liquid substrates of all types;

FIG. 2 illustrates a 5 μm breadth by 8 μm height cell of a colonyshowing an ovoid cell containing nine round hydrocarbon vesicles, adictyosome, a nucleolus, and a parietal chloroplast;

FIG. 3 is a microphotograph of a green hydrocarbon-rich colony showingovoid protuberant cells, each containing five and more round hydrocarbonvesicles. At the perimeter are seven hydrocarbon droplets extruded fromthe colony by pressure from the glass cover slip; and

FIG. 4 is an exemplary schematic diagram of a hydrocarbon productionprocess of the present invention.

DETAILED DESCRIPTION OF THE INVENTION Assay

Previous investigations (Ohmori, et al. 1984; and Wolf, et al, 1985)verified that colonies containing greater than 20% of dry weight asbotryococcenoid hydrocarbons were buoyant and that colonies containingless than 5% hydrocarbons remained sunken at the bottom of the culturevessel, providing a visual method for selecting cells withcommercial-grade hydrocarbons. FIG. 1 shows floating colonies ofBotryococcus sp. with a corresponding 30% hydrocarbon content.Additionally, the hydrocarbon-vesicle counts within cells, as drawn inFIG. 2, were found to be in direct correlation to hydrocarbon yields,providing microscopy for confirming selection of cells and colonies withhigh hydrocarbon contents. Colonies in hydrocarbon-enhancement wereplaced in the dark without supplemental carbon dioxide gas to eliminateartifacts of flotation from bubbles. Environmental conditions were 150μE/m²/sec PAR to 1700 μE/m²/sec PAR at 25-37° Centigrade. Nutrientchemicals were reagent grade in all laboratory-scale experiments. Infield-scale tests, fertilizer grade chemical nutrients were provided. Inorder to minimize or eliminate ammonia, nitrates were optionally kilndried prior to admixture. Supplementation with 0.1% to 100% carbondioxide gas or carbonate (e.g., 10 mg/ml sodium bicarbonate or potassiumbicarbonate) increased hydrocarbon synthesis substantially, especially,when under the highest light intensities with soluble elements of thepresent invention.

Clones of Botryococcus sp. originated from the collections from Nature.Axenic clones, from the Ninsei strain disclosed in provisionalapplication Ser. No. 60/678,711, filed May 6, 2005 and incorporatedherein by reference, micropropagated rapidly in light on novel carbon-and nutrient-supplemented solid media. Solids included selections from0.5% to 1.5% agar, colloids, gelatin, plastic gels, cellulose, plantfibers, synthetic fibers, polymers, woven fabrics, nonwovens, paper,broadcloth, iron, stainless steel, netting, moist glass, brick,concrete, plastic, foams, nylon, and ceramic surfaces. Sunken colonieswith very low hydrocarbon content were archived as controls in Showanutrients, US Patent Plant 6169, and are incorporated by referenceherein, to provide controls in experiments. Up to the point ofexperimentation, the varieties were variously maintained in Showa media;Chu 13 (Chu 1942); other conventional phycological nutrient media; andwere supplemented with trace minerals, spring water, or soil-waterextracts in aqueous solution. Botryococcus sp. was maintained in definediron-, zinc- and phosphate-enriched media of the present invention.

Colonies of Ninsei are variably-shaped groups of cells held together inthe cups of tough sporopollenin-like matrices. Depth of color depends onthe light regime, density or culture and physiological state of thecolonies. All color designations are made with reference to the MunsellBook of Color. Normal healthy colonies range from 5 GY 7/8 to 2.5 GY8/12 on the Munsell color chart and these Ninsei colonies, fullypigmented with chlorophylls, may float at the surface of growingcultures with high hydrocarbon content that may reflect goldenovertones.

The Ninsei variety is characterized at an average green hue of 2.5 GY6/10±50% on the Munsell color chart for healthy colonies. In contrast,Showa is described as a yellow of 2.5 Y 7/8 on the Munsell color chart.Vegetative reproduction resulting in increased colony count ismaintained as long as there is chlorophyll content to reflect 2.5 GYhues. No growth has been observed in the Y through YR Munsell Colorrange, but conversion of carbon stores to hydrocarbon may continue upinto the YR brown state of decline. Flotation is concomitant with growthof botryococcenes branched hydrocarbons (C_(n)H_(2n-10), n=30-37). Whenreleased from the colonial matrix, cells of the colonies are 5 μm to 10μm spheres often pressed by neighboring cells into irregular shapes.Within the colony, the cells are wedged into an almond-shape betweenneighboring cells. Deposits of hydrocarbon, 0.1 μm to 1 μm in diameter,are present in the cytoplasm, wall, and matrix. An occasional cell ofNinsei exhibits a depression at the outer tip of the cell that mostfrequently appears in cells with few hydrocarbon vesicles and may be aresult of secretion of oils. The name of the strain is, in fact, derivedfrom the urn shape of cells, reminiscent of shapes of large ceramicwares by the artist of Kyoto, ca. 1600 AD, Ninsei.

The colonial unit is spherical and aggregates of units contribute to theformation of irregular grape-like clusters observed in large colonies.During rapid growth of the novel strain, colonies are generally smallerthan Showa's 50 μm colonies. In Ninsei, smaller colonies may range from10 μm to 45 μm in diameter. Colonies of 100 or more cells arepredominantly composed of irregularly shaped units that fragment intoroughly rounded colonies. Ninsei is visually distinguishable from otherstrains of the variety by its deep green hue, small size attributable torapid growth, cell structure, and niche. Botryococcus braunii var.Showa, strain Ninsei was deposited at the American Type CultureCollection (Post Office Box 1549, Manassas, Va. 20108) in March 2006 andassigned ATCC No. PTA-7441.

Solution culture nutrient concentrations meant for vascular plant crops,widely recognized by crop nutritionists for the past half century asdefined hydroponic fertilizer formulae for flowering plants, had notbeen previously applied to Botryococcus species. Generally, hydroponicculture media effectively suppress protistans by being far too highlyconcentrated for unicellular organisms. In contrast, for the presentinvention, Botryococcus species were found to grow in full-strengthHydroponic Solutions and, to the complete surprise of the inventor, innutrient salt concentrations previously thought to be toxic to plantsand to other life. For the present invention, major modifications weremade to substantially increase zinc, potassium, calcium, magnesium,manganese, iron, and phosphate concentrations well beyond those of theconventional phycological and hydroponic solutions. Phosphate saltsprovided the additional benefit of buffering within a range of pH 6 topH 7. Furthermore, concentrations of soluble zinc ions (Zn⁺²) wereincreased one hundred times to two thousand times that of finalconcentrations found for soluble zinc in vegetable crop production; andsoluble iron was increased up to quadruple the concentration used invegetable solution culture. For example, in hydroponics the followingfinal concentrations of nutrient elements has been widely regarded bythose in the field for the successful solution culture of tomato crops:“119 ppm N; 30 ppm P; 140 ppm K; 100 ppm Ca; 24 ppm Mg; 32 ppm S; 2.5ppm Fe; 0.25 ppm B; 0.25 ppm Mn; 0.025 ppm Zn; 0.01 ppm Cu; and 0.005ppm Mo,” (Growing Plants in Solution Culture, in Hawkes, G. R. et al,editors, (1980), Western Fertilizer Handbook, The Interstate Printersand Publishers, Inc., Danville, Ill. Pages 185-193).

The preferred nutrient enrichment solution of the present invention ishereinafter denoted as KwiK (see Table below). Botryococcus sp. wasrapidly propagated in KwiK and it should be noted that the preferredmild formulation of KwiK contains over fifty times the total phosphatesand many times higher Zn⁺² concentrations than Chu 13 (Table ComparingMedia, below) and hydroponic media.

The present invention comprises trials with TrebouxiophyceaenBotryococcus species; hereinafter Botryococcus sp. The preferredenvironment for maintenance of colonies required buffering byappropriate concentrations of nutrient phosphate salts and carbonateadjusted to pH 6.3 to pH 6.8, preferably to pH 6.7. Colonies ofBotryococcus sp. floated within hours when supported by the ZiP mediumof the present invention, containing 80 mM to 150 mM total phosphatesand up to 70 ppm Zn⁺², in a range between pH 7.0 to pH 8.3, and underhigh light intensity, and it was, thereupon, hypothesized that theorganism prefers the surface niche where moisture, illumination, andhigh salt concentrations abound.

EXPERIMENTAL

Methods, compositions and systems of the present invention provide meansfor in vitro growth of transport fuel hydrocarbons.

Stock cultures were taken from bottom-dwelling colonies that had beenmaintained in conventional liquid culture media, typified byapproximately 1 ppm to 10 ppm phosphate, 0.01 ppm to 0.3 ppm iron, and0.01 ppm to 0.5 ppm zinc, as for example, in Showa medium and otherphycological media. Aqueous nutrient solutions were placed in sterileculture tubes and flasks and steam sterilized twice for 60 minutes.Following inoculation and growth, colonies were concentrated withovernight settling and approximately one million submerged greencolonies were collected from the bottom of culture vessels. Equalvolumes were resuspended into replicate culture vessels with equalvolumes of enhancement media in the highest light intensities availablein preparation for experiments. Control cultures were transferred intoequal volumes of Showa medium and placed side by side under identicalconditions as controls.

In water with up to 300 mM phosphates, bottom-dwelling colonies began torise to the surface within 5 hours. After two weeks or more in 300 mMphosphate, the colonies remained floating, but cleared to amber. Byanalysis of a subtractive matrix, eliminating individual compounds fromeach application, greater than 80 mM total phosphates, hundreds of timesthe concentration of phosphate salts of conventional phycological media,stimulated the rise of colonies to the surface, most rapidly when in thepresence of soluble ions of Mg, Mn, Fe, and Zn⁺². The same buoyantresponse of all of the colonies occurred in the presence ofcorrespondingly high concentrations of balanced equimolar phosphatesalts, regardless of the counter-ions whether they were selected fromammonium, potassium, magnesium, or sodium. It was also important tobalance acidic with basic phosphate salts at equimolar concentrationsbecause a 2 mM difference was found to exceed the pH-tolerance ofphycological specimens.

With further testing and modifications of the highly concentrated saltsto reflect nitrogen-phosphoric-potash, N—P—K, typical of hydroponicnutrients, and with chelated secondary and trace minerals, the preferredbuffered enrichment solution with 80 mM to 120 mM potassium phosphatewas developed for the present invention. The solution was adjusted tofall within a range of about pH 6.5 to pH 7 by regulation ofapproximately equimolar mono- and di-potassium phosphates (MKP and DKP)or other phosphate salts. Phosphates were selected because of thehigh-energy requirement of adenosine triphosphate, ATP, for metabolismof cellular resources into hydrocarbons. Potassium was selected as thecounter-ion of choice because it is a major nutrient that does notprecipitate at high concentrations. In the present invention,supplementation with 1 ppm to 90 ppm soluble Zn⁺² was critical toacceleration of hydrocarbon chain elongation and 0.1 ppm to 10 ppmsoluble iron was essential to photosynthesis. That is, in illuminatedcultures in KwiK supplemented preferably with 5 ppm soluble zinc andiron, colonies grew hydrocarbons at an accelerated rate by provision ofa controlled upwelling of Kwik power. Therefore, trace minerals weremodified by replacing the mineral salts with chelated salts at 0.03 ppmto 50 ppm concentrations for each element in order to maintainsolubility in the presence of high concentrations of phosphate. In thecourse of the present invention, rapid hydrocarbon production by greencolonies was maintained in the presence of KwiK elements with the mosthighly preferred concentration of 5 ppm to 9 ppm soluble iron.Enrichment by Zn⁺² and phosphate in a second nutrient solution was foundto enhance hydrocarbon content of the colonies while providing abiologically competitive advantage to the colonies of the presentinvention. The enriched solution for accelerated hydrocarbon productionis, hereinafter, denoted ZiP. In the process development of Botryococcussp., colonies from populations floating above the meniscus of ZiP werevisually verified with high hydrocarbon-vesicle counts, harvested andtransferred to ZiP to ripen with hydrocarbons.

Maintenance of cultures for long durations under high light intensityillumination ranging from 500 to 1700 μE/m²/sec PAR, 8-20 h light, at25-35° C. is preferred in KwiK supplemented with saturated carbondioxide or bicarbonate, especially when under the highest lightintensities. Alternatively, carbonated water with ZiP may be metered into maintain rapidly growing hydrocarbons. The buoyant colonies of thepresent invention were characterized by high growth content, upwards of5% to 50% dry weight of mixed lipids. The preferred environment formaintenance of the floating colonies requires buffering by appropriateconcentrations of available 20 mM to 125 mM phosphates, 3 ppm to 10 ppmFe, and 0.1 ppm to 70 ppm Zn⁺², where 80 mM to 90 mM phosphates with 0.2ppm to 45 ppm chelated Zn⁺² is preferred. Colonies tolerate a broadphysiological range from pH 5.5 up to pH 8.3; however, under high lightintensity and with carbonate availability, continuous adjustment tomaintain pH 6.8 is essential to maintain the solubility of minerals inhigh concentrations of phosphate. Organic substrates for enhancement ofhydrocarbons include 1 mm to 100 mM Krebs Cycle carboxylates, preferablywith citrate as an acid component of citrate-phosphate buffer;mevalonates; methionines, preferably adenosyl-methionine; alcohols; andfatty acids. For rapid growth, at concentrations above 1 mM totalphosphates, it is important to prevent precipitation, especially bycalcium and magnesium, by addition of appropriate concentrations ofsequestering agents such as disodium-, diammonium-, anddipotassium-ethylenediaminetetraacetates; citrate; carboxylates; and thelike. Additionally, maintenance of acidic environments assists withsolubility of media with high concentrations of phosphate.Agriculturally accepted sources of Zn⁺² include, without exclusion ofany other zinc salts, zinc sulfate, zinc oxide, zinc carbonate, zincchloride, zinc citrate, zinc oxysulfate, zinc ammonium sulfate, and zincnitrate, supplemented by chelation with, for example, salts of EDTA,HEEDTA, NTA, DTPA, EDDHA, and the like. Commercially available 6% to 14%Zn⁺² as diammonium EDTA may be alkaline which may be compensated byaddition of the monobasic phosphate salt to adjust the final solution topH 7. Sources of iron include, without exclusion of any other ironsupplements, iron sulfate, iron oxide, iron filings, ferric chloride,ferric ammonium citrates, ferrous salts, soil extracts, and supplementedby chelation with, for example, salts of EDTA, HEEDTA, NTA, DTPA, EDDHA,and the like. The most highly preferred medium eliminates all sources ofammoniacal nitrogen in order to fully enhance hydrocarbon production ofmass cultures. It is the hypothesis of the present invention thathydrocarbon synthesis may be fully optimized by providing nutrientsbeneficial to photosynthesis, including 50 ppm to 200 ppm magnesium aspart of the chlorophyll molecule and about 5 ppm to 10 ppm soluble ironthat is essential to electron transport. Preferably, in for example KwiKor ZiP, inclusion of 7 ppm to 9 ppm soluble ferric or ferrous ions inthe media accomplishes the same when balanced equally by 0.2 ppm solubleMn, and with provision of high light intensity illumination, carbondioxide gas, and 0.2 ppm to 50 ppm Zn⁺², the synthesis of hydrocarbonsmay be optimized.

For a population of Botryococcus colonies, 2 mM and greaterconcentrations of phosphates, 500 ppm to 1200 ppm nitrate salt, 500 ppmpotassium salt, 3 ppm to 10 ppm Fe, 0.1 ppm to 3 ppm Mn, and 0.1 ppm to5 ppm Zn⁺² are required for long-term growth of hydrocarbons. Therecommended and preferred upper limits are 120 mM total phosphates at pH7 and 50 ppm soluble zinc. For hydrocarbon synthesis, supplementationswith 25 ppm to 250 ppm soluble magnesium, 0.2 ppm soluble manganese, 5-9ppm soluble iron, and 0.1 ppm to 50 ppm Zn⁺², are preferred. Tracemetals are preferably chelated. Most preferably, for maintenance of thegrowth of hydrocarbons, the medium is supplemented as specified in KwiK.The preferred method for making ZiP is to mix and sterilize a solutionof 160 mM to 400 mM total phosphates and add equal volumes of thephosphate solution to pre-sterilized KwiK resulting in 80 mM to 200 mMtotal phosphates ZiP solutions with chelated nutrients. The preferredZiP solution at 88 mM to 150 mM balanced phosphates with 2 ppm to 50 ppmZn⁺² and with 10 ppm to 20 ppm Fe in KwiK supports growth ofhydrocarbons.

The biosynthesis of hydrocarbons is an energy-intensive pathway that maybe accelerated by the availability of very high concentrations offerrous, ferric, Zn⁺² and phosphates of the present invention. Thus, theenzymes in this system require phosphate-energy-complexes, such as theZn⁺²-requiring farnesyl pyrophosphate synthase, as demonstrated in thecurrent invention. As the photosynthetic organism also responds rapidlyto the uppermost concentrations of phosphates, commercialbatch-processing of colonies is envisioned, whilst continuous processingof partial populations in KwiK is an open option. Notably, exposure to200 mM to 300 mM phosphates and to 100 ppm Zn⁺² resulted in a colorchange of the colonies toward amber over the long duration ofapproximately two to eight weeks, implicating rapid batch processing asthe method of choice.

KwiK Medium, adjusted to pH 7 with phosphate buffer Concentration KwiKComponent Range Preferred KH₂PO₄ 80-800 ppm 136 ppm K₂HPO₄ 80-1000 ppm174 ppm KNO₃ 500-2500 ppm 570 ppm Chelants 80-1500 ppm 200-1000 ppmMgSO₄ 1-1000 ppm 100 ppm Ca⁺² 1-800 ppm 88 ppm Fe 0.3-20 ppm 5 ppm to 10ppm Mn 0.1-3 ppm 0.2 ppm Cu 0.01-0.1 ppm 0.01 ppm B 0.2-2 ppm 0.2 ppmZn⁺² 0.3-50 ppm 2 ppm Mo 0.001-0.05 ppm 0.02 ppm Co 0.001-0.05 ppm 0.002ppm

ZiP medium, adjusted to pH 7 with phosphate buffers Concentration ZiPComponent Range Preferred Phosphates 0.1% to 3% 0.5% to 0.8% Zn⁺² 0.2ppm to 70 ppm 36 ppm to 50 ppm

Organic ZiP Medium in Spring Water ZiP Component ConcentrationSupplement Range Preferred Citrate-Phosphate Buffer 0.1% to 3% 0.8% to2% Zn⁺² 0.2 ppm to 70 ppm 36 ppm to 50 ppm Potassium acetate 0.1% to 3%0.5% to 1% Comparisons of Nutrient Media Nutrient Salt KwiK ZiP Chu 13KH₂PO₄ 272 ppm 1.4% 0 K₂HPO₄ 348 ppm 1.7% 0.001% KNO₃ 570 ppm 0.05% 0.005% Zn⁺²  2 ppm 50 ppm 0.5 ppm

Colonies of Botryococcus sp. were found to live and grow at the surfaceof the water or on moist substrates, whereas, other general methods ofculture involved immersion in water. Exceedingly slow growth has beenobserved in the Y through YR Munsell Color range and conversion ofcarbon stores to hydrocarbon may continue up into the amber state ofdecline without appropriate elements. Colonies of cells are heldtogether by a matrix that is rich in hydrocarbons. As coloniesaccumulate hydrocarbons, they exhibit a correspondingly higherabsorbance of ultraviolet light, as measured spectrophotometrically.This UV-absorbance characteristic of the present invention may beapplied to cell-sorting instrumentation attuned to selection of coloniesand cells with maximized hydrocarbon content. The colonial unit isspherical and aggregates of units contribute to the irregulargrape-cluster formations observed in large colonies of the genericnamesake from Latin, Botryococcus.

Colonies grow particularly well in KwiK-supplemented water-moistenedsolid media under continuous or periodic (e.g., 16:8 h LD) PAR lightexposure, with high light intensities up to direct sunlight as high as500 to 1700 μE/m²/sec in high density cultures and temperatures of 20°to 37°.

Defined growth media for the strains of the present invention includeprimary, secondary and trace metal plant nutrients. The most highlypreferred formula provides balanced N—P—K at many times theconcentrations of conventional nutrients. Balanced formulations includenitrate, phosphate and potash sources of fertilizers at rates exceedinghydroponics of flowering plants; as well as the secondary nutrients,Ca⁺², S, and Mg; and soluble micronutrients such as ions of Fe, Mn,Zn⁺², Cu, B, Mo, Co, and Ni. Through matrix analyses in the course ofthe present invention, it was found that supplementation with a cocktailof metal ions is preferred for maintenance of growth of hydrocarbons andpreferably include 5 mM to 25 mM magnesium, 0.1 ppm to 3 ppm manganese,3 ppm to 10 ppm iron, and 0.01 mM to 0.1 mM Zn⁺². Total phosphates maybe in the range from 2 mM to 150 mM phosphates. Sources of typicalsolution culture nutrients are, for example, selected from myriad andvarious available compounds as generally accepted and known by those inthe art.

The present invention elucidates the only process for which the growthof transport fuel hydrocarbons in Botryococcus may be undertaken byfilling a chemical niche of KwiK and ZiP components. Such high Fe, Zn⁺²and phosphate concentrations have otherwise proven detrimental to lowerphotosynthetic organism as evidenced by the low concentrations ofconventional formulations (as for example in Chu 1942; and US PatentPlant 6169). Botryococcus sp. produces chemical structures ofC_(n)H_(2n-10), n=30-37; C₂₅ to C₃₁ n-alkadienes and trienes; C₄₀H₇₈;carotenoids; and fatty acids; and isomers thereof.

Botryococcus sp. may have adapted to the chemical extremes of exclusiveconcentrations of phosphate, up to 3% in vitro, and as high ascrystalline in Nature. Zinc and manganese have long histories of beingformulated into human medications for their germ-fighting benefits and,thus, very high concentrations of Mn and Zn⁺² provide clear competitiveadvantages for Botryococcus sp. to survive where other microorganismsdie. Flotation enables it to be transported to live at the surface oredge of the exclusive moist solid medium. Botryococcus sp. occupies thedefined niche of the present invention. The preponderance of mixedhydrocarbons, when accumulated in high cellular concentrations, functionas naturally effective ultraviolet sunlight blocking agents, necessaryfor survival on land; and as such, the whole organism as well as itsextract may be utilized in topical sun block formulae.

Example 1

Hydrological shear forces are greatest at the air:water interface. Whenthe liquid cultures of Botryococcus sp. in ZiP were placed in a shakertable (>100 rpm) cloaked in 3% carbon dioxide, oil was pressed out ofthe colonies by shearing forces. When brought to a stand still, the oilfloated at the surface to be harvested by skimming.

In healthy sunlit water-borne cultures, colonies rose off the bottom tovarious levels up to the top half of the column in ZiP culturessupplemented with 36 ppm to 50 ppm Zn⁺², 120 mM phosphate and KwiKcomponents at about pH 7. All control colonies in conventionalphycological media dropped below the bottom half of the culture tubewithin an hour. After residing in the dark for 12 hours, colonies in ZiPremained floating at the meniscus, while, in contrast, the colonies ofcontrol cultures remained sunken at the bottom of the culture vessels.Starting from colonies maintained for a week in KwiK, the time toflotation of the population was approximately 1 day after exposure toZiP. Rate of flotation was affected by species selection. Notably, thepreferred formulation of ZiP contains over 300 times the phosphate and100 times the zinc concentrations of Chu 13 (Table Comparing Media,above).

Example 2

The process system of the present invention is schematically depicted inFIG. 4, wherein the mud niche is mimicked by provision of a continuouslymoistened solid medium such as a fabric beltway that is sufficientlytight in its weave to prevent the colonies from slipping through. Forexample, 25 to 50 micron Nitex® Broadcloth is an appropriate selection.Nitex® Broadcloth 10 microns to 600 microns is the material of choicefor plankton nets. The fabric belt is inoculated with Botryococcus spp.and growth is maintained by continuous misting with carbon dioxidegas-supplemented KwiK and natural solar illumination. Initially,gas-carbonation assists by sustaining acidity that prevents loss ofmetallic nutrients to precipitation. Different oleomic strains,varieties and species may be interspersed in the culture. Whensufficient biomass is measured by achieving growth to 1 to 10 mm depth,the nutrient mist is replaced with a 10 mM bicarbonate-supplemented ZiPmist. Over time, bicarbonate raises the alkalinity of the nutrientsolution, thus, provision of an oleomic environment is dependent uponmetering appropriate buffering agents into the culture environment tomaintain solubility of nutrients. The colonies are allowed sufficienttime to ripen by visually monitoring hydrocarbon-vesicle counts withinlive cells taken through random samplings. At the determined time ofmaximum vesicle count exceeding 8 hydrocarbon-vesicles per cell in agiven plane, as per FIG. 2, hydrocarbons are harvested from live cellsby applying aqueous solutions or phytobland organic solvents as 30 PSIto 100 PSI pressurized misting sprays for 1 to 45 minutes. Applyingpressure to exude hydrocarbons from cells was photographically recordedin FIG. 3, where exogenous droplets of oil droplets were visuallyobserved in vivo. Preferably, pressurized water is applied to the matfollowing a design that supplies sufficient hydrological shear to presshydrocarbons out of the colonies while keeping the cells alive;therefore, the least pressure that forces exudation of oils is preferredand is dependent on the thickness of the mat. The cycle is repeateduntil the inoculum is exhausted.

The formulations and methods of the present invention may be applied tovirtually any variety of living organism that metabolizes hydrocarbons,most preferably photosynthetic organisms. These photosynthetic organismsinclude protistans, bacteria, and plants. Plants include innumerableagricultural and horticultural species and varieties, known arts tothose in the field.

The methods of the present invention are amenable to batch processing ofcaptive hydrocarbon vesicles. Sheared botryococcenes allow thepossibility of the continuous harvest of products. Thus, industrialmimicry of Nature's competitive advantage represents an improvement onsystems suited to the production and harvest of renewable hydrocarbons.Botryococcenes are the natural product of choice as a starting materialfor a number of hydrocarbon based products, such as petrochemicals,pharmaceuticals, and fuels.

1. A microorganism designated Ninsei, a representative of which havingbeen deposited under ATCC Accession No. PTA-744.
 2. A substantially pureculture of Ninsei, having the ATCC Accession No. PTA-7441.
 3. A nutrientcomposition comprising: 80-800 ppm K₂PO₄, 80-1000 ppm K₂HPO₄, 500-2500ppm KNO₃, 80-800 ppm Chelants, 1-1000 ppm MgSO₄, 1-800 ppm Ca⁺², 3-10ppm Fe, 0.1-3 ppm Mn, 0.01-0.1 ppm Cu, 0.2-2 ppm B, 0.3-50 ppm Zn²⁺,0.001-0.05 ppm Mo, 0.001-0.05 ppm Co.
 4. The nutrient composition ofclaim 3, comprising about 136 ppm KH₂PO₄, about 17 ppm K₂HPO₄, 570 ppmKNO₃, about 200-1750 ppm chelants, about 100 ppm MgSO₄, about 88 ppmCa⁺², at least about 5 ppm Fe, about 0.2 ppm Mn, about 0.01 ppm Cu,about 0.2 ppm B, about 0.2 ppm Zn²⁺, about 0.02 ppm Mo, and about 0.002ppm Co.
 5. The nutrient composition of claim 3 in liquid water andculture of Botryococcus sp. therein.